PROJECT SUMMARY There is a fundamental gap in our knowledge whether natural mechanisms protect high-risk children from caries and arise from microbial interactions of commensal bacteria in the oral cavity, or from the interplay of microbiome and tooth, mediated by saliva. The long-term goal is to prevent early childhood caries (ECC) in North American Indigenous children through protective treatments that may also be applicable in the broader population. The central hypothesis, based on the research team’s strong preliminary data, is that children with and without ECC differ in one or more key drivers: i) microbially reduced acidogenicity of S. mutans, or ii) enamel and dentin properties, composition, or biochemical fingerprint. The objective in this application is to integrate observational and basic science, from associations to experiments that test underlying caries protection mechanisms in children with high levels of S. mutans. The study rationale is based on strong evidence that i) Rothia sp. actively control S. mutans acidogenesis, and ii) enamel and dentin differ in composition between the two groups, with lead, cadmium, and sulfur lower in teeth from caries-free children compared with the unaffected region of caries-affected teeth. The research team plans to pursue the following three Specific Aims: Aim 1. Test whether and how Rothia and/or other oral species may mitigate the cariogenic effects of acidogenic bacteria. Aim 2. Test whether and how tooth properties modulate the susceptibility to acid dissolution of enamel and dentin. Aim 3. Test how tooth substrate or saliva affect acidogenicity and spatial structure of biofilms, and whether spatial structure of biofilms grown from ex vivo dental plaque differs between ECC-affected and CF children. The contribution is expected to achieve high impact by going beyond single-risk factor studies to investigate caries-protective mechanisms involving microbial genetics, biofilm organization and tooth composition. The proposed research is innovative, because we shift focus to the small percentage of Indigenous children with high loads of S. mutans and without caries history, use state-of-the art imaging techniques of in vitro controlled biofilm growth on standardized enamel chips, and integrate multimodal analyses of enamel and dentin properties, biochemical fingerprint, and mineral composition. This contribution will be significant because dental caries disproportionally affects North American Indigenous children. After successful completion of this project, new mechanistic insights into molecular interactions and physiological functions of commensal oral flora to reduce acid production in cariogenic species can inform new caries preventive therapeutic strategies.